Inversion of Thr and Ile Side Chain Stereochemistry in a ProteinMolecule: Impact on the Folding Stability and Structure of the ShK ToxinProtein Molecule

摘要

ShK toxin is a cysteine-rich 35 residue protein ion channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the impact of inverting the side chain stereochemistry of individual Thr and Ile residues on the properties of the ShK protein molecule. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side chain stereochemistry of individual Thr/Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogs each containing either an allo-Thr or an allo-Ile residue. The three allo-Thr/allo-Ile containing ShK polypeptides were each able to fold into a defined protein product, but with different folding propensities. Relative thermal stabilities of these ShK analogs were measured and were in agreement with the MD simulation data. Structures of the three ShK analogs were determined by quasi-racemic X-ray crystallography and were similar to wild type ShK protein. All three ShK analogs retained Kv1.3 ion channel blocking activity.